NK-cell function is regulated by the integration of signals received from activating and inhibitory receptors. Here we show that a novel immune receptor, T-cell Ig and mucin-containing domain-3 (Tim-3), is expressed on resting human NK cells and is up-regulated on activation. The NK92 NK-cell line engineered to overexpress Tim-3 showed a marked increase in IFN-␥ production in the presence of soluble rhGal-9 or Raji tumor cells engineered to express Gal-9. The Tim-3 ؉ population of low-dose IL-12/IL-18-activated primary NK cells significantly increased IFN-␥ production in response to soluble rhGal-9, Gal-9 presented by cell lines, and primary acute myelogenous leukemia (AML) targets that endogenously express Gal-9. This effect is highly specific as Tim-3 Ab blockade significantly decreased IFN-␥ production, and Tim-3 cross-linking induced ERK activation and degradation of IB␣. Exposure to Gal-9-expressing target cells had little effect on CD107a degranulation. Reconstituted NK cells obtained from patients after hematopoietic cell transplantation had diminished expression of Tim-3 compared with paired donors. This observation correlates with the known IFN-␥ defect seen early posttransplantation. In conclusion, we show that Tim-3 functions as a human NK-cell coreceptor to enhance IFN-␥ production, which has important implications for control of infectious disease and cancer. (Blood. 2012;119(13): 3064-3072) IntroductionHuman NK cells are lymphocytes that develop from hematopoietic progenitor cells in the BM and secondary lymphoid tissues. 1 Peripheral blood (PB) NK cells are phenotypically defined as expressing the surface receptor CD56 (neural cell adhesion molecule [NCAM]) and lacking expression of CD3. 2 They mediate their function through the exocytosis of lytic granules that contain perforin and granzymes, 3 the expression of death receptor ligands, 4 the expression of FcR␥III, which mediates Ab-dependent cellmediated cytotoxicity, 5 and the secretion of cytokines and chemokines. 6 As a result, NK cells take part in both the innate and adaptive immune systems and play important roles in the control of viral infections, pregnancy, tumor immunosurveillance, and hematopoietic cell transplantation (HCT). 7,8 The ability of NK cells to differentiate normal healthy cells (self) from virally infected or malignantly transformed cells (nonself) is regulated by a sophisticated repertoire of cell-surface receptors that control their activation, proliferation, and effector functions. [9][10][11] Recently, a novel receptor, T-cell Ig and mucincontaining domain-3 (Tim-3), has been described to have various roles in immune regulation and is highly expressed on NK cells in mice and humans. [12][13][14][15][16][17] Tim-3 is a type I membrane glycoprotein that was first identified as a cell marker of terminally differentiated CD4 ϩ Th1 cells. 18 Galectin-9 (Gal-9), a 40-kDa S-type ␤-galactoside binding lectin, is a known ligand for Tim-3 and is highly expressed in tissues of the immune system, such as the BM, lymph nodes, thymu...

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This study evaluates the mechanism by which bispecific and trispecific killer cell engagers (BiKEs and TriKEs) act to trigger human NK cell effector function and investigates their ability to induce NK cell cytokine and chemokine production against human B-cell leukemia. We examined the ability of BiKEs and TriKEs to trigger NK cell activation through direct CD16 signaling, measuring intracellular Ca2+ mobilization, secretion of lytic granules, induction of target cell apoptosis and production of cytokine and chemokines in response to the Raji cell line and primary leukemia targets. Resting NK cells triggered by the recombinant reagents led to intracellular Ca2+ mobilization through direct CD16 signaling. Co-culture of reagent-treated resting NK cells with Raji targets resulted in significant increases in NK cell degranulation and target cell death. BiKEs and TriKEs effectively mediated NK cytotoxicity of Raji targets at high and low effector-to-target (E:T) ratios and maintained functional stability after 24 and 48 hours of culture in human serum. NK cell production of IFN-γ, TNF-α, GM-CSF, IL-8, MIP-1α and RANTES was differentially induced in the presence of recombinant reagents and Raji targets. Moreover, significant increases in NK cell degranulation and enhancement of IFN-γ production against primary ALL and CLL targets were induced with reagent treatment of resting NK cells. In conclusion, BiKEs and TriKEs directly trigger NK cell activation through CD16, significantly increasing NK cell cytolytic activity and cytokine production against tumor targets, demonstrating their therapeutic potential for enhancing NK cell immunotherapies for leukemias and lymphomas.

Key Points NK cells and their expression of FcRγIII (CD16) are decreased in MDS and inversely correlate with a substantial increase in MDSCs. CD16xCD33 BiKE potently activates blood and marrow MDS-NK cells at all diseases stages to lyse CD33+ MDS and CD33+ MDSC targets.

Purpose The graft versus leukemia (GVL) effect by Natural Killer (NK) cells prevents relapse following hematopoietic stem cell transplantation. We determined whether a novel bi-specific killer cell engager (BiKE) signaling through CD16 and targeting CD33 could activate NK cells at high potency against AML targets. Experimental Design We investigated the ability of our fully humanized CD16x33 BiKE to trigger in vitro NK cell activation against HL60 (CD33+), RAJI (CD33−), and primary AML targets (de novo, refractory and post transplant) to determine whether treatment with CD16x33 BiKE in combination with an ADAM17 inhibitor could prevent CD16 shedding (a novel inhibitory mechanism induced by NK cell activation) and overcome inhibition of class I MHC recognizing inhibitory receptors. Results NK cell cytotoxicity and cytokine release were specifically triggered by the CD16x33 BiKE when cells were cultured with HL60 targets, CD33+ de novo and refractory AML targets. Combination treatment with CD16x33 BiKE and ADAM17 inhibitor resulted in inhibition of CD16 shedding in NK cells, and enhanced NK cell activation. Treatment of NK cells from double umbilical cord blood transplant (UCBT) recipients with the CD16x33 BiKE resulted in activation, especially in those recipients with CMV reactivation. Conclusion CD16x33 BiKE can overcome self inhibitory signals and effectively elicit NK cell effector activity against AML. These in vitro studies highlight the potential of CD16x33 BiKE ± ADAM17 inhibition to enhance NK cell activation and specificity against CD33+ AML, which optimally could be applied in patients with relapsed AML or for adjuvant anti-leukemic therapy post-transplantation.

Although NK cell alloreactivity has been dominated by studies of KIR, we hypothesized that NKG2A and LIR-1, present on 53±13% and 36±18% of normal NK cells, plays a role in NK cell killing of primary leukemia targets. KIR− cells, which comprise nearly half of the circulating NK cell population, exhibited tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both AML and ALL targets could be rendered susceptible to lysis by fresh resting KIR− NK cells when inhibitory receptor-MHC class I interactions were blocked by pan-HLA antibodies demonstrating that these cells were functionally competent. Blockade of a single inhibitory receptor resulted in slight increases in killing, while combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR− NK cells showing that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR− NK cells is determined by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemia.

Background Natural killer (NK) cells are potent cytotoxic lymphocytes that play a critical role in tumor immunosurveillance and control. Cancer stem cells (CSC) initiate and sustain tumor cell growth, mediate drug refractory cancer relapse and express the well-known surface marker CD133. Methods DNA fragments from two fully humanized single chain fragment variable (scFv) antibody recognizing CD16 on NK-cells and CD133 on CSC were genetically spliced forming a novel drug, 16 × 133 BiKE that simultaneously recognizes these antigen to facilitate an immunologic synapse. The anti-CD133 was created using a fusion protein prepared by fusing DNA fragments encoding the two extracellular domains of CD133. Immunization of mice with the resulting fusion protein generated an unique antibody that recognized the molecular framework and was species cross-reactive. Results In vitro 51chromium release cytotoxicity assays at both high and low effector:target ratios demonstrated the ability of the heterodimeric biological drug to greatly enhance NK-cell killing of human Caco-2 colorectal carcinoma cells known to overexpress CD133. The tumor associated antigen specificity of the drug for CD133 even enhanced NK-cell cytotoxicity against the NK-resistant human Burkitt's lymphoma Daudi cell line, which has less than 5% CD133 surface expression. Flow cytometry analysis revealed increases in NK-cell degranulation and Interferon-γ production upon co-culture with Caco-2 targets in the presence of the drug. Conclusion These studies demonstrate that the innate immune system can be effectively recruited to kill CSC using bispecific antibodies targeting CD133, and that this anti-CD133 scFv may be useful in this bispecific platform or, perhaps, in the design of more complex trispecific molecules for carcinoma therapy.

A heterodimeric bispecific biological recombinant drug was synthesized by splicing DNA fragments from two fully humanized single-chain variable-fragment (scFV) antibody fragments forming a novel drug simultaneously recognizing the CD16 natural killer (NK) cell marker and the cancer marker epithelial cell adhesion molecule (EpCAM). The drug precipitously enhanced the killing of human carcinomas of the prostate, breast, colon, head, and neck even at very low effector:target ratios. The drug EpCAM16 rendered even nonactivated NK cellproficient killers and activated them to kill via degranulation and cytokine production. Studies show that bispecific antibodies can be used to induce proficient killing of the carcinoma targets that ordinarily are resistant to NK-mediated killing. Apparently, the innate immune system can be effectively recruited to kill cancer cells using the bispecific antibody platform and EpCAM targeting.